QUINONE DERIVATIVES FOR USE IN THE MODULATION OF REDOX STATUS OF INDIVIDUALS
Methods of modulating, adjusting, and maintenance of the glutathione redox status of an individual, or cell, tissues, bodily fluids, or body compartments of the individual, are disclosed, as are compositions suitable for such modulating, adjusting, and maintenance. The modulation or adjustment is achieved by altering the amounts of reduced glutathione versus oxidized glutathione in the individual, or cells, tissues, bodily fluids, or body compartments of the individual. Modulation, adjustment, and maintenance of the redox status of an individual enables treatment, prevention or suppression of diseases or symptoms associated with diseases. These methods are achieved by using quinone derivatives.
This application claims priority benefit of U.S. Provisional Patent Application No. 61/698,431 filed Sep. 7, 2012, and of U.S. Provisional Patent Application No. 61/792,797 filed Mar. 15, 2013. The entire contents of those applications are hereby incorporated by reference herein.
TECHNICAL FIELD OF THE INVENTIONThe application discloses compositions and methods useful for modulation of the glutathione redox status and redox potential of an individual, useful in treatment, prevention, or suppression of diseases, by administering redox-active compounds (such as tocotrienol quinones, for example, alpha-tocotrienol quinone).
BACKGROUNDA proper balance between oxidation and reduction reactions in cells, tissues, and organisms is vital for health. Maintenance of such a proper redox balance is, for example, critical for proper functioning of processes such as glycolysis, the citric acid cycle, and oxidative phosphorylation, which depend upon electron-transfer reactions in order to produce ATP, the major energy carrier in living organisms.
Several diseases are associated with disrupted redox processes in cells, tissues, or organisms. Atkuri et al. (Proc. Nat'l. Acad. Sci. USA, 106(10):3941 (2009)) showed that patients with disorders affecting mitochondrial function suffered from systemic oxidative stress. However, the appropriate treatment for diseases involving oxidative stress is by no means clear. Jones (Antioxid. Redox Signal. 8(9&10):1865 (2006)) notes that administration of antioxidants demonstrates mixed results in improving health.
The current invention provides compositions and methods for modulating, adjusting, and maintaining the redox status of an individual at an appropriate level to improve or enhance the health of the individual.
SUMMARY OF THE INVENTIONThe invention provides compounds and methods to address the common glutathione cycle defect associated with several diseases, including mitochondrial and neurodegenerative diseases. Specifically, the compounds and methods regenerate reduced glutathione and increase the glutathione charge couple, resulting in: i) restoration of redox balance and metabolic control; ii) reduction in the generation of reactive oxygen species; and iii) arrest and reversal of disease.
The methods of the invention encompass modulating, adjusting, and maintaining a healthy glutathione redox potential in an individual, or in the cells, tissues, organs, or bodily fluids of an individual, by altering the ratios of reduced glutathione (GSH) to oxidized glutathione (GSSG) in the individual, or in the cells, tissues, organs, or bodily fluids of the individual.
The present invention comprises multiple aspects, features and embodiments, where such multiple aspects, features and embodiments can be combined and permuted in any desired manner. These and other aspects, features and embodiments of the present invention will become evident upon reference to the remainder of this application, including the following detailed description. In addition, various references are set forth herein that describe in more detail certain compositions, and/or methods; all such references are incorporated herein by reference in their entirety.
The invention embraces compositions and methods for modulating, adjusting, and maintaining the redox status of an individual at an appropriate level to improve or enhance the health of the individual, or to treat, prevent, or suppress certain diseases or symptoms of certain diseases.
By “subject,” “individual,” or “patient” is meant an individual organism, preferably a vertebrate, more preferably a mammal, most preferably a human.
“Treating” a disease with the compounds and methods discussed herein is defined as administering one or more of the compounds discussed herein, with or without additional therapeutic agents, in order to reduce or eliminate either the disease or one or more symptoms of the disease, or to retard the progression of the disease or of one or more symptoms of the disease, or to reduce the severity of the disease or of one or more symptoms of the disease. “Suppression” of a disease with the compounds and methods discussed herein is defined as administering one or more of the compounds discussed herein, with or without additional therapeutic agents, in order to suppress the clinical manifestation of the disease, or to suppress the manifestation of adverse symptoms of the disease. The distinction between treatment and suppression is that treatment occurs after adverse symptoms of the disease are manifest in a subject, while suppression occurs before adverse symptoms of the disease are manifest in a subject. Suppression may be partial, substantially total, or total. Because many of the mitochondrial disorders are inherited, genetic screening can be used to identify patients at risk of the disease. The compounds and methods of the invention can then be administered to asymptomatic patients at risk of developing the clinical symptoms of the disease, in order to suppress the appearance of any adverse symptoms. “Therapeutic use” of the compounds discussed herein is defined as using one or more of the compounds discussed herein to treat or suppress a disease, as defined above. A “therapeutically effective amount” of a compound is an amount of a compound which, when administered to a subject, is sufficient to reduce or eliminate either one or more symptoms of a disease, or to retard the progression of one or more symptoms of a disease, or to reduce the severity of one or more symptoms of a disease, or to suppress the clinical manifestation of a disease, or to suppress the manifestation of adverse symptoms of a disease. A therapeutically effective amount can be given in one or more administrations. An “effective amount” of a compound embraces both a therapeutically effective amount, as well as an amount effective to modulate, normalize, or enhance one or more energy biomarkers in a subject.
“Modulation” of, or to “modulate,” an energy biomarker means to change the level of the energy biomarker towards a desired value, or to change the level of the energy biomarker in a desired direction (e.g., increase or decrease). Modulation can include, but is not limited to, normalization and enhancement as defined below.
“Normalization” of, or to “normalize,” an energy biomarker is defined as changing the level of the energy biomarker from a pathological value towards a normal value, where the normal value of the energy biomarker can be 1) the level of the energy biomarker in a healthy person or subject, or 2) a level of the energy biomarker that alleviates one or more undesirable symptoms in the person or subject. That is, to normalize an energy biomarker which is depressed in a disease state means to increase the level of the energy biomarker towards the normal (healthy) value or towards a value which alleviates an undesirable symptom; to normalize an energy biomarker which is elevated in a disease state means to decrease the level of the energy biomarker towards the normal (healthy) value or towards a value which alleviates an undesirable symptom.
“Enhancement” of, or to “enhance,” energy biomarkers means to intentionally change the level of one or more energy biomarkers away from either the normal value, or the value before enhancement, in order to achieve a beneficial or desired effect. For example, in a situation where significant energy demands are placed on a subject, it may be desirable to increase the level of ATP in that subject to a level above the normal level of ATP in that subject. Enhancement can also be of beneficial effect in a subject suffering from a disease or pathology such as a mitochondrial disease, in that normalizing an energy biomarker may not achieve the optimum outcome for the subject; in such cases, enhancement of one or more energy biomarkers can be beneficial, for example, higher-than-normal levels of ATP, or lower-than-normal levels of lactic acid (lactate) can be beneficial to such a subject.
While the compounds described herein can occur and can be used as the neutral (non-salt) compound, the description is intended to embrace all salts of the compounds described herein, as well as methods of using such salts of the compounds. In one embodiment, the salts of the compounds comprise pharmaceutically acceptable salts. Pharmaceutically acceptable salts are those salts which can be administered as drugs or pharmaceuticals to humans and/or animals and which, upon administration, retain at least some of the biological activity of the free compound (neutral compound or non-salt compound). The desired salt of a basic compound may be prepared by methods known to those of skill in the art by treating the compound with an acid. Examples of inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Examples of organic acids include, but are not limited to, formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, sulfonic acids, and salicylic acid. Salts of basic compounds with amino acids, such as aspartate salts and glutamate salts, can also be prepared. The desired salt of an acidic compound can be prepared by methods known to those of skill in the art by treating the compound with a base. Examples of inorganic salts of acid compounds include, but are not limited to, alkali metal and alkaline earth salts, such as sodium salts, potassium salts, magnesium salts, and calcium salts; ammonium salts; and aluminum salts. Examples of organic salts of acid compounds include, but are not limited to, procaine, dibenzylamine, N-ethylpiperidine, N,N′-dibenzylethylenediamine, and triethylamine salts. Salts of acidic compounds with amino acids, such as lysine salts, can also be prepared. Several pharmaceutically acceptable salts are disclosed in Berge, J. Pharm. Sci. 66:1 (1977).
The invention also includes all stereoisomers and geometric isomers of the compounds, including diastereomers, enantiomers, and cis/trans (E/Z) isomers. The invention also includes mixtures of stereoisomers and/or geometric isomers in any ratio, including, but not limited to, racemic mixtures. Unless stereochemistry is explicitly indicated in a structure, the structure is intended to embrace all possible stereoisomers of the compound depicted. If stereochemistry is explicitly indicated for one portion or portions of a molecule, but not for another portion or portions of a molecule, the structure is intended to embrace all possible stereoisomers for the portion or portions where stereochemistry is not explicitly indicated.
The description of compounds herein also includes all isotopologues, for example, partially deuterated or perdeuterated analogs of all compounds herein.
The compounds can be administered in prodrug form. Prodrugs are derivatives of the compounds which are themselves relatively inactive, but which convert into the active compound when introduced into the subject in which they are used, by a chemical or biological process in vivo, such as an enzymatic conversion. Suitable prodrug formulations include, but are not limited to, peptide conjugates of the compounds of the invention and esters of compounds of the inventions. Further discussion of suitable prodrugs is provided in H. Bundgaard, Design of Prodrugs, New York: Elsevier, 1985; in R. Silverman, The Organic Chemistry of Drug Design and Drug Action, Boston: Elsevier, 2004; in R. L. Juliano (ed.), Biological Approaches to the Controlled Delivery of Drugs (Annals of the New York Academy of Sciences, v. 507), New York: N.Y. Academy of Sciences, 1987; and in E. B. Roche (ed.), Design of Biopharmaceutical Properties Through Prodrugs and Analogs (Symposium sponsored by Medicinal Chemistry Section, APhA Academy of Pharmaceutical Sciences, November 1976 national meeting, Orlando, Fla.), Washington: The Academy, 1977.
The various compounds of the invention can be administered either as therapeutic agents in and of themselves, or as prodrugs which will convert to other therapeutically effective or effective substances in the body.
The term “alkyl” refers to saturated aliphatic groups including straight-chain, branched-chain, cyclic groups, and combinations thereof, having the number of carbon atoms specified, or if no number is specified, having up to 12 carbon atoms. “Straight-chain alkyl” or “linear alkyl” group refers to alkyl groups that are neither cyclic nor branched, commonly designated as “n-alkyl” groups. One subset of alkyl groups is —(C1-C6)alkyl which include groups such as methyl, ethyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, n-pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and any other alkyl group containing between one and five carbon atoms, where the —(C1-C6)alkyl groups can be attached via any valence on the —(C1-C6) alkyl groups.
GlutathioneGluthathione is the molecule (2S)-2-amino-5-[[(2R)-1-(carboxymethylamino)-1-oxo-3-sulfanylpropan-2-yl]amino]-5-oxopentanoic acid (IUPAC), and has the following structure:
which can be described as the tripeptide gamma-Glu-Cys-Gly. Glutathione is commonly abbreviated as GSH. The oxidized form of glutathione is the disulfide-bridged dimeric form of the tripeptide, and is abbreviated as GSSG. Glutathione can also form mixed disulfides with cysteine-containing proteins; the glutathione-protein molecule is abbreviated as GS-Pro. Glutathione can also form persulfides by reaction with hydrogen sulfide or sulfane sulfur. Glutathione persulfide is abbreviated as GSSH. Glutathione is an example of an energy biomarker that can be modulated, normalized, or enhanced using the methods of the invention and the compounds disclosed herein, and which can be modulated, normalized, or enhanced to adjust the redox status of an individual in accordance with the invention.
Compounds for Use in Modulating, Normalizing, or Enhancing Redox Status of an IndividualIn one embodiment, compounds of Formula I are useful in the invention for modulating glutathione levels:
where R is selected from:
where the asterisk * indicates the attachment of R in Formula I; where R1, R2, and R3 are independently H or C1-C6 alkyl, m is an integer from 0 to 9 inclusive, and the bonds indicated by a dashed line can be either double or single bonds. In some embodiments m is selected from 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, m is selected from 1, 2, or 3. In some embodiments, m is 2. In another embodiment, R1, R2, and R3 are CH3. In another embodiment, m is 2 and R1, R2, and R3 are CH3.
In one embodiment, compounds of Formula I-ox and Formula I-red are useful in the invention for modulating glutathione levels:
where R1, R2, and R3 are independently H or C1-C6 alkyl, m is an integer from 0 to 9 inclusive, and the bonds indicated by a dashed line can be either double or single bonds. In some embodiments m is selected from 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, m is selected from 1, 2, or 3. In some embodiments, m is 2. In another embodiment, R1, R2, and R3 are CH3. In another embodiment, m is 2 and R1, R2, and R3 are CH3.
In another embodiment, compounds of Formula I-D-ox and Formula I-D-red are useful in the invention for modulating glutathione levels:
where R1, R2, and R3 are independently H or C1-C6 alkyl, and m is an integer from 0 to 9 inclusive. In some embodiments m is selected from 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, m is selected from 1, 2, or 3. In some embodiments, m is 2. In another embodiment, R1, R2, and R3 are CH3. In another embodiment, m is 2 and R1, R2, and R3 are CH3.
In another embodiment, compounds of Formula I-S-ox and Formula I-S-red are useful in the invention for modulating glutathione levels:
where R1, R2, and R3 are independently H or C1-C6 alkyl, and m is an integer from 0 to 9 inclusive. In some embodiments m is selected from 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, m is selected from 1, 2, or 3. In some embodiments, m is 2. In another embodiment, R1, R2, and R3 are CH3. In another embodiment, m is 2 and R1, R2, and R3 are CH3.
In another embodiment, the compounds of Formula I-ox that are useful in the invention for modulating glutathione levels are tocotrienol quinones of the following structures:
including stereoisomers and salts thereof.
In other embodiments, the compound of Formula I-ox is a tocotrienol quinone. In other embodiments, the compound of Formula I-ox is alpha-tocotrienol quinone. In other embodiments, the compound of Formula I-ox is beta-tocotrienol quinone. In other embodiments, the compound of Formula I-ox is gamma-tocotrienol quinone. In other embodiments, the compound of Formula I-ox is delta-tocotrienol quinone.
In other embodiments, the compounds of Formula I-ox used to modulate glutathione levels comprise an effective amount of a mixture of two or more tocotrienol quinones selected from alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone and delta-tocotrienol quinone.
In another embodiment, the compounds of Formula I-red that are useful in the invention for modulating glutathione levels are tocotrienol hydroquinones of the following structures:
including stereoisomers and salts thereof.
In other embodiments, the compound of Formula I-red is a tocotrienol hydroquinone. In other embodiments, the compound of Formula I-red is alpha-tocotrienol hydroquinone. In other embodiments, the compound of Formula I-red is beta-tocotrienol hydroquinone. In other embodiments, the compound of Formula I-red is gamma-tocotrienol hydroquinone. In other embodiments, the compound of Formula I-red is delta-tocotrienol hydroquinone.
In other embodiments, the compounds of Formula I-red used to modulate glutathione levels comprise an effective amount of a mixture of two or more tocotrienol hydroquinones selected from alpha-tocotrienol hydroquinone, beta-tocotrienol hydroquinone, gamma-tocotrienol hydroquinone and delta-tocotrienol hydroquinone.
In another embodiment, the compounds of Formula I-ox that are useful in the invention for modulating glutathione levels are tocopherol quinones of the following structures:
including stereoisomers and salts thereof.
In other embodiments, the compound of Formula I-ox is a tocopherol quinone. In other embodiments, the compound of Formula I-ox is alpha-tocopherol quinone. In other embodiments, the compound of Formula I-ox is beta-tocopherol quinone. In other embodiments, the compound of Formula I-ox is gamma-tocopherol quinone. In other embodiments, the compound of Formula I-ox is delta-tocopherol quinone.
In other embodiments, the compounds of Formula I-ox used to modulate glutathione levels comprise an effective amount of a mixture of two or more tocopherol quinones selected from alpha-tocopherol quinone, beta-tocopherol quinone, gamma-tocopherol quinone and delta-tocopherol quinone.
In another embodiment, the compounds of Formula I-red that are useful in the invention for modulating glutathione levels are tocopherol hydroquinones of the following structures:
including stereoisomers and salts thereof.
In other embodiments, the compound of Formula I-red is a tocopherol hydroquinone. In other embodiments, the compound of Formula I-red is alpha-tocopherol hydroquinone. In other embodiments, the compound of Formula I-red is beta-tocopherol hydroquinone. In other embodiments, the compound of Formula I-red is gamma-tocopherol hydroquinone. In other embodiments, the compound of Formula I-red is delta-tocopherol hydroquinone.
In other embodiments, the compounds of Formula I-red used to modulate glutathione levels comprise an effective amount of a mixture of two or more tocopherol hydroquinones selected from alpha-tocopherol hydroquinone, beta-tocopherol hydroquinone, gamma-tocopherol hydroquinone and delta-tocopherol hydroquinone.
The quinones described herein, such as tocotrienol quinones and tocopherol quinones, can be used in their oxidized form (Formula I-ox, Formula I-D-ox, Formula I-S-ox), or can be used in their reduced hydroquinone form (Formula I-red, Formula I-D-red, Formula I-S-red). The quinone (cyclohexadienedione) form and hydroquinone (benzenediol) form are readily interconverted with appropriate reagents. The quinone form can be treated in a biphasic mixture of an ethereal solvent with a basic aqueous solution of Na2S2O4 (Vogel, A. I. et al. Vogel's Textbook of Practical Organic Chemistry, 5th Edition, Prentice Hall: New York, 1996; Section 9.6.14 Quinones, “Reduction to the Hydroquinone”). Standard workup in the absence of oxygen yields the desired hydroquinone form. The hydroquinone form can be oxidized to the quinone form with oxidizing agents such as ceric ammonium nitrate (CAN) or ferric chloride. The quinone and hydroquinone forms are also readily interconverted electrochemically, as is well known in the art. See, e.g., Section 33.4 of Streitweiser & Heathcock, Introduction to Organic Chemistry, New York: Macmillan, 1976.
In another embodiment, compounds of Formula II are useful in the invention for modulating glutathione levels:
where R′ is selected from
wherein R21 and R22 are independently of each other hydrogen, —(C1-C6)alkyl or —O—(C1-C6)alkyl; or R21 and R22 together represent —CH═CH—CH═CH—;
R23 is (C1-C6)alkyl;
X is —CH═CH— or —C≡C—;
m is 1-10; n is 1-5; k is 1-3, with the proviso that when k is an integer of 2 to 3, n is optionally variable from 1-5 in each occurrence of the —X—(CH2)n— group;
Y is —OR24, —CN or —COOR25; and
R24 and R25 are independently of each other selected from hydrogen, —CN, —(C1-C6)alkyl, —(C1-C6)haloalkyl; —C(═O)—(C1-C6)alkyl; —C(═O)—(C1-C6)haloalkyl; —C(═O)—NH(C1-C6)alkyl; —C(═O)—N((C1-C6)alkyl)2 and —C(═O)—NH2; or any stereoisomer, mixture of stereoisomers, prodrug, metabolite, salt, crystalline form, non-crystalline form, hydrate or solvate thereof.
In another embodiment, R21, R22, and R23 are independently selected from —(C1-C6)alkyl. In another embodiment, R21 and R22 are independently selected from —O(C1-C6)alkyl. In another embodiment, R21 and R22 are hydrogen and R23 is —(C1-C6)alkyl. In another embodiment, R21, R22 and R23 are hydrogen. In another embodiment, R21 and R22 together represent —CH═CH—CH═CH—. In another embodiment, X is —CH═CH—. In another embodiment, X is —C≡C—. In another embodiment, Y is —OR24. In another embodiment Y is —CN or —COOR25. In another embodiment R24 is hydrogen, —C(═O)—(C1-C6)alkyl, or —C(═O)—(C1-C6)haloalkyl. In another embodiment R25 is hydrogen or —(C1-C6)alkyl. In another embodiment, m is 1-5. In another embodiment, m is 2-5. In another embodiment n is 1-4. In another embodiment, n is 1-3. In another embodiment n is 1-2. In another embodiment, k is 2-3.
In another embodiment, R21, R22 and R23 are —CH3; X is —CH═CH—; m is 4; n is 1-3; k is 1-2; and Y is —OH. In another embodiment, R21, R22 and R23 are CH3; X is —C≡C—; m is 4; n is 1-3; k is 1-2; and Y is —OH. In another embodiment, R21 and R22 together represent —CH═CH—CH═CH—; R23 is CH3; X is —CH═CH—; m is 4; n is 1-3, k is 1-2; and Y is —OH. In another embodiment, R21 and R22 together represent —CH═CH—CH═CH—; R23 is CH3; X is —C≡C—; m is 4; n is 3, k is 1-3 and Y is —OH.
In another embodiment, compounds of Formula II-ox or Formula II-red are useful in the invention for modulating glutathione levels:
wherein R21 and R22 are independently of each other hydrogen, —(C1-C6)alkyl or —O—(C1-C6)alkyl; or R21 and R22 together represent —CH═CH—CH═CH—;
R23 is (C1-C6)alkyl;
X is —CH═CH— or —C≡C—;
m is 1-10; n is 1-5; k is 1-3, with the proviso that when k is an integer of 2 to 3, n is optionally variable from 1-5 in each occurrence of the —X—(CH2)n— group;
Y is —OR24, —CN or —COOR25; and
R24 and R25 are independently of each other selected from hydrogen, —CN, —(C1-C6)alkyl, —(C1-C6)haloalkyl; —C(═O)—(C1-C6)alkyl; —C(═O)—(C1-C6)haloalkyl; —C(═O)—NH(C1-C6)alkyl; —C(═O)—N((C1-C6)alkyl)2 and —C(═O)—NH2; or any stereoisomer, mixture of stereoisomers, prodrug, metabolite, salt, crystalline form, non-crystalline form, hydrate or solvate thereof.
In another embodiment, R21, R22, and R23 are independently selected from —(C1-C6)alkyl. In another embodiment, R21 and R22 are independently selected from —O(C1-C6)alkyl. In another embodiment, R21 and R22 are hydrogen and R23 is —(C1-C6)alkyl. In another embodiment, R21, R22 and R23 are hydrogen. In another embodiment, R21 and R22 together represent —CH═CH—CH═CH—. In another embodiment, X is —CH═CH—. In another embodiment, X is —C≡C—. In another embodiment, Y is —OR24. In another embodiment Y is —CN or —COOR25. In another embodiment R24 is hydrogen, —C(═O)—(C1-C6)alkyl, or —C(═O)—(C1-C6)haloalkyl. In another embodiment R25 is hydrogen or —(C1-C6)alkyl. In another embodiment, m is 1-5. In another embodiment, m is 2-5. In another embodiment n is 1-4. In another embodiment, n is 1-3. In another embodiment n is 1-2. In another embodiment, k is 2-3.
In another embodiment, R21, R22 and R23 are —CH3; X is —CH═CH—; m is 4; n is 1-3; k is 1-2; and Y is —OH. In another embodiment, R21, R22 and R23 are CH3; X is —C≡C—; m is 4; n is 1-3; k is 1-2; and Y is —OH. In another embodiment, R21 and R22 together represent —CH═CH—CH═CH—; R23 is CH3; X is —CH═CH—; m is 4; n is 1-3, k is 1-2; and Y is —OH. In another embodiment, R21 and R22 together represent —CH═CH—CH═CH—; R23 is CH3; X is —C≡C—; m is 4; n is 3, k is 1-3 and Y is —OH.
In another embodiment the compound of Formula II for modulating glutathione levels includes, but is not limited to:
- 2-(12-hydroxydodeca-5,8-dien-1-yl)-3,5,6-trimethylcyclohexa-2,5-diene-1,4-dione;
- 2-(10-hydroxydeca-5,8-diyn-1-yl)-3,5,6-trimethylcyclohexa-2,5-diene-1,4-dione;
- 2-(12-hydroxydodeca-5,8-diyn-1-yl)-3,5,6-trimethylcyclohexa-2,5-diene-1,4-dione;
- 2-(10-hydroxydeca-5,8-dien-1-yl)-3,5,6-trimethylcyclohexa-2,5-diene-1,4-dione;
- 2-(9-hydroxynon-5-yn-1-yl)-3,5,6-trimethylcyclohexa-2,5-diene-1,4-dione;
- 2-(11-hydroxyundeca-5,8-diyn-1-yl)-3,5,6-trimethylcyclohexa-2,5-diene-1,4-dione;
- 13-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)trideca-5,8-diynenitrile;
- 13-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)trideca-5,8-diynoic acid;
- 13-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)trideca-5,8-dienenitrile;
- 13-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)trideca-5,8-dienoic acid;
- N,N-dimethyl-13-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)trideca-5,8-diynamide;
- 13-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)trideca-5,8-diynamide;
- 2-(12-hydroxydodeca-5,8-diyn-1-yl)-3-methylnaphthalene-1,4-dione;
- 2-(11-hydroxyundeca-5,8-diyn-1-yl)-3-methylnaphthalene-1,4-dione;
- 2-(10-hydroxydeca-5,8-diyn-1-yl)-3-methylnaphthalene-1,4-dione;
- 2-(10-hydroxydeca-5,8-dien-1-yl)-3-methylnaphthalene-1,4-dione;
- 2-(10-hydroxydeca-5,8-dien-1-yl)-3-methylnaphthalene-1,4-dione;
- 2-(10-hydroxydeca-5,8-dien-1-yl)-3-methylnaphthalene-1,4-dione;
or any stereoisomer, mixture of stereoisomers, prodrug, metabolite, salt, crystalline form, non-crystalline form, hydrate or solvate thereof.
In another embodiment, the invention embraces 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone or any stereoisomer, mixture of stereoisomers, prodrug, metabolite, salt, crystalline form, non-crystalline form, hydrate or solvate thereof, for use in modulating glutathione levels. 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone, (CAS Registry number 80809-81-9) is also known as Docebenone, AA861 or aa-861, and has the structure:
In another embodiment, the invention embraces the lactone; 2,3,5-trimethyl-6-(2-(2-methyl-5-oxotetrahydrofuran-2-yl)ethyl)cyclohexa-2,5-diene-1,4-dione (CAS Registry numbers 3121-68-4, 15716-16-2, 22625-17-8, 816456-29-8):
and its open chain carboxylic acid 4-hydroxy-4-methyl-6-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)hexanoic acid (CAS Registry number 1948-76-1):
or any stereoisomer, mixture of stereoisomers, prodrug, metabolite, salt, crystalline form, non-crystalline form, hydrate or solvate thereof, for use in modulating glutathione levels.
Synthesis of CompoundsThe compounds described herein can be readily synthesized by a variety of methods known in the art. The syntheses of the compounds of Formula II described herein are detailed in, for example, U.S. Pat. No. 4,393,075 hereby incorporated by reference in its entirety. 2,3,5-Trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone can be purchased from Sigma-Aldrich, St. Louis, Mo. (catalog number A3711, CAS Registry Number 80809-81-0, under the name AA-861 or 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-p-benzoquinone).
In Vitro and In Vivo Assessment of Efficacy of Compounds: Measurement of Glutathione Ratios and Redox Potential of Glutathione CoupleMeasurement of the redox potential of the glutathione pool provides a snapshot of the redox balance of the cell, tissue, or organism in which the potential is measured.
The glutathione potential of a sample can be determined using the Nernst equation:
E=Eo−(RT/nF)(ln([GSSG]/[GSH]2))
where Eo is the redox potential of the glutathione couple under standard conditions, R is the gas constant, T is the absolute temperature (that is, in degrees Kelvin, K), n is the number of electrons transferred in the reaction, and F is Faraday's constant. [GSSG] represents the concentration of oxidized glutathione, while [GSH] is the concentration of reduced glutathione. See, for example, Jones, Dean P., “Redox Potential of GSH/GSSG Couple: Assay and Biological Significance” in Methods of Enzymology: Protein Sensors and Reactive Oxygen Species—Part B: Thiol Enzymes and Proteins (Sies, H. and Packer, L., editors), Vol. 348, pp. 93-112 (2002); and Jonas et al., American Journal of Clinical Nutrition, 72:181-189 (2000).
Since glutathione redox potentials in individuals are negative, between −170 mV and −90 mV, changes in the glutathione redox potential are indicated by the absolute value of the change and the direction of the change. Thus, if a subject has a glutathione redox potential of −120 mV, a change in redox potential of an absolute value 10 mV more negative indicates that the redox potential of the subject has changed to −130 mV. A change in glutathione redox potential of an absolute value of at least about 10 mV more negative indicates that the absolute value of the change is greater than 10, and the change is more negative. If the individual's starting glutathione redox potential was −120 mV, then an example of a change in redox potential of an absolute value of at least about 10 mV more negative would be a change of the glutathione redox potential to −135 mV; the absolute value of the change is 15, which is at least about the absolute value of 10, and the change to the potential is in the negative direction.
Glutathione concentration can be measured using a variety of methods, such as that described by Pastore, A., et al. “Fully automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine in plasma and urine,” Clin. Chem. (Washington, D.C.) 44:825-832 (1998) (see Example 1 below). Other glutathione assays have been described in Serru et al., “Quantification of Reduced and Oxidized Glutathione in Whole Blood Samples by Capillary Electrophoresis,” Clin. Chem. (Washington, D.C.) 47:1321-1324 (2001); Giustarini et al., “An improved HPLC measurement for GSH and GSSG in human blood,” Free Radic. Biol. Med. 35:1365-1372 (2003); Harfield et al., “Electrochemical determination of glutathione: a review,” Analyst 137(10):2285-96 (2012); Yap et al., “Determination of GSH, GSSG, and GSNO using HPLC with electrochemical detection,” Methods Enzymol. 473:137-47 (2010); and Monostori et al., “Determination of glutathione and glutathione disulfide in biological samples: an in-depth review,” J Chromatogr B Analyt Technol Biomed Life Sci. 877(28):3331-46 (2009).
Plasma levels of GSH, GSSG, Cys, and CySS can be used to calculate the in vivo Eh values. Samples are collected using the procedure of Jones et al (2009 Free Radical Biology & Medicine 47(10):1329-1338). This method uses bromobimane to alkylate free thiols and HPLC and either electrochemical or MSMS to separate, detect, and quantify the molecules. One method of analyzing the most common monothiols and disulfides (cystine, cysteine, reduced (GSH) and oxidized glutathione (GSSG)) present in human plasma is to use bathophenanthroline disulfonic acid as the internal standard (IS), with complete separation of all the target analytes and IS at 35° C. on a C18 RP column (250 mm×4.6 mm, 3 micron) achieved using 0.2% TFA:Acetonitrile as a mobile phase pumped at the rate of 0.6 ml per minute using an electrochemical detector in DC mode at the detector potential of 1475 mV. The protocol detailed below in the examples can also be used for HPLC analysis of thiols.
Target Range of Glutathione Redox PotentialA “subject having a glutathione redox potential disorder,” “individual having a glutathione redox potential disorder,” or “patient having a glutathione redox potential disorder,” is a subject, individual, or patient having a glutathione redox potential lying outside the normal range for a healthy age-matched individual. The range of glutathione redox potentials compatible with life is roughly −170 millivolts to −90 millivolts in human plasma. Comparison of the glutathione potential of an individual to a healthy age-matched individual serving as control will indicate whether the potential is at a pathological value. Comparison can be made to a healthy age-matched, sex-matched individual who has the same smoking status as the person being tested (that is, smokers should be compared to healthy age-matched, sex-matched smokers; non-smokers should be compared to healthy age-matched, sex-matched non-smokers).
In another embodiment, the comparison can be made to an average value over a group of control individuals who are matched (by age, sex, and smoking status) to the test individual. A group of ten, fifty, or one hundred people can be used. Using a group for comparison will allow calculation of a standard deviation for the control group, and the test individual can be considered for therapy (that is, considered as having a glutathione redox potential disorder) if the subject's glutathione redox potential is at least one standard deviation, at least two standard deviations, or at least three standard deviations away from the mean value of the control group. Preferably, the standard deviation of the group of control subjects is no more than +/−5 mV.
If an individual undergoes therapy by virtue of having a glutathione redox potential which is at least one standard deviation, at least two standard deviations, or at least three standard deviations away from the mean value, they can undergo therapy according to the invention until their glutathione redox potential is within no more than three standard deviations, two standard deviations, or one standard deviation of the mean value of the control group.
In some embodiments, the individual can undergo therapy to shift their glutathione redox potential to an absolute value of about 10 mV, about 20 mV, about 30 mV, or about 40 mV more negative relative to their glutathione redox potential prior to undergoing therapy. In some embodiments, the individual can undergo therapy to shift their glutathione redox potential to an absolute value of about at least 10 mV, about at least 20 mV, about at least 30 mV, or about at least 40 mV more negative relative to their glutathione redox potential prior to undergoing therapy.
Examples of values for healthy individuals in plasma would be about −156 mV at age 34, −149 mV at age 49, and −140 mV at age 59. For persons aged 30 to 40 years, an example range is −160 mV to −150 mV. For persons aged 40 to 50 years, an example range is −150 mV to −140 mV. For persons aged 50 to 60 years, an example range is −140 mV to −120 mV. Other ranges can be used as target ranges depending on other specific characteristics of the control group, such as body mass index, hematocrit, cholesterol levels, or other biomarkers or clinical test results.
Glutathione levels are preferably measured in plasma, but can also be measured in whole blood, blood serum, cerebrospinal fluid, semen, breast milk, umbilical cord blood, and tissues or tissue homogenates such as umbilical cord tissue and skin biopsy.
Diseases and Symptoms Amenable to TreatmentSeveral diseases and symptoms can be treated or suppressed using the invention. Addressing the underlying redox imbalance provides a broad mechanism for alleviating several pathological metabolic processes.
Mitochondrial diseases are one type of disease that can be treated by modulation of glutathione redox potential. These diseases include Myoclonic Epilepsy with Ragged Red Fibers (MERRF); Mitochondrial Myopathy, Encephalopathy, Lactacidosis, and Stroke (MELAS); Leber's Hereditary Optic Neuropathy (LHON); Dominant Optic Atrophy (DOA); Chronic Progressive External Ophthalmoplegia (CPEO); Leigh Disease (Leigh Syndrome); Leigh-like Syndrome; Kearns-Sayre Syndrome (KSS); Friedreich's Ataxia (FA); Mitochondrial Neurogastrointestinal Encephalopathy disease (MNGIE); Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP); Spinocerebellar Ataxia (SCA), also called Machado-Joseph disease; overlap syndromes; Co-Enzyme Q10 (CoQ10) Deficiency; Complex I deficiency; Complex II deficiency; Complex III deficiency; Complex IV deficiency; and Complex V deficiency.
Organic acidurias (organic acidemias) can also be treated by modulation of glutathione redox potential. Methylmalonic aciduria (methylmalonic acidemia, MMA), isovaleric aciduria (isovaleric acidemia; IVA), or other organic acidurias can be treated using the compounds and methods described herein.
Ataxia-telangiectasia (A-T) (also known as Boder-Sedgwick syndrome or Louis-Bar syndrome), and Ataxia-telangiectasia-like disorder (ATLD) can also be treated by modulation of glutathione redox potential, by using the compounds and methods described herein.
There are also several diseases which, while not usually categorized primarily as redox dysfunction disorders or oxidative stress disorders, are exacerbated by, or have significant involvement from, redox potential imbalance and/or oxidative stress. These include diseases beyond the “classic” mitochondrial diseases that display significant mitochondrial dysfunction. Among such diseases are cardiomyopathy; encephalomyopathy; renal tubular acidosis; neurodegenerative diseases (Johnson et al., Nutrients 4:1399-1440 (2012)); Parkinson's disease (Sian et al., Ann. Neurol. 36:348-355 (1994)); Alzheimer's disease; amyotrophic lateral sclerosis (ALS); epilepsy; Huntington's Disease; schizophrenia; bipolar disorder; aging and age-associated diseases (Kretzschmar et al., Sports Medicine 15(3)196-209 (1993); Samiec et al., Free Radical Biology and Medicine 24(5):699-704 (1998); Sekhar et al., American Journal of Clinical Nutrition, 94:847-853 (2011)); macular degeneration (Samiec et al., Free Radical Biology and Medicine 24(5):699-704 (1998)); diabetes (Samiec et al., Free Radical Biology and Medicine 24(5):699-704 (1998)); cerebrovascular accidents, such as ischemic stroke and hemorrhagic stroke; certain cancers which display mitochondrial dysfunction; and cystic fibrosis (Roum et al., J. Appl. Physiol. 75(6):2419-2424 (1993)). Alteration of glutathione redox potential also occurs in persons undergoing chemotherapy (Jonas et al., American Journal of Clinical Nutrition 72:181-189 (2000)).
Thus, in one embodiment of the invention, a subject having a glutathione redox potential disorder, who also manifests one of the diseases above, can be treated according to the invention in order to modulate the redox status of the subject, in order to treat the disease.
Leigh Syndrome and Modulation of Glutathione PotentialExample 2 demonstrates the correlation between modulation of the glutathione redox potential and improvement in clinical measures of Leigh syndrome (Leigh disease), an illness characterized by redox dysfunction and oxidative stress. Leigh syndrome is a rare, fatal inherited neurodegenerative disorder with an incidence of 1 in 40,000, and which predominantly affects children. Leigh syndrome is characterized by an overproduction of reactive oxygen species (ROS) and decompensation of the glutathione cycle.
Example 2 describes a subject-controlled Phase 2 clinical trial of alpha-tocotrienol quinone in genetically confirmed Leigh syndrome subjects ranging in ages from one to 13, spanning seven distinct and defined nDNA or mtDNA mutations. All subjects were treated for three months with 100 mg of alpha-tocotrienol quinone orally administered, three times daily. No drug related adverse events were recorded. All children exhibited arrest and reversal of clinical disease progression regardless of age, genetic mutation or disease severity. The primary endpoints of the study were the Newcastle Pediatric Mitochondrial Disease Scale (NPMDS), the Gross Motor Function Measure (GMFM) and PedsQL Neuromuscular Module (PedsQL). All demonstrated statistically significant improvement. Sections 1-3 (organ system function) of the NPMDS improved by a mean of 7.1 (p=0.005). Section 4 of the NPMDS (quality of life) scores improved an average of 4.6 (p=0.01). Consistent with section 4 of the NPMDS, there was also a statistically significant improvement in the mean PedsQL of 14.6 (p=0.03). The GMFM recorded a mean improvement of 8.9 (p=0.01). In addition, all children had an improvement of one class on the Movement Disorder-Childhood Rating Scale (MD-CRS)—the secondary endpoint of the study. Glutathione cycle biomarkers were measured as an indicator of disease pathology and drug action. All patients showed a statistically significant increase in reduced glutathione derived from isolated lymphocytes from 8.5 to 25.1 nmol/mg cell protein (p=0.002) and a reduction in oxidized glutathione to 0.25 nmol/mg cell protein (p=0.005).
Alpha-tocotrienol quinone is a small-molecule therapeutic which can ameliorate reduced glutathione levels through NQO1-catalyzed electron transfer from NADPH (Enns, G. M., et al., Mol. Genet. Metab. 105, 91-102 (2012); Shrader, W. D., et al., Bioorg. Med. Chem. Lett. 21, 3693-3698 (2011)). By increasing reduced glutathione, alpha-tocotrienol quinone prevents ROS-mediated cell injury and death. Alpha-tocotrienol quinone has been recently reported to be safe and effective in the treatment of several inherited mitochondrial diseases (Enns, G. M., et al., Mol. Genet. Metab. 105, 91-102 (2012); Sadun, A. A., et al., Arch Neurol 69, 331-338 (2012); Blankenberg, F., et al., Ann. Neurol. 70, S148-S148 (2011)).
A total of 10 children with genetically confirmed Leigh syndrome were enrolled in the study and treated with alpha-tocotrienol quinone for at least 3 months. Baseline patient characteristics, including the specific genetic mutation, are shown in Table 1 (
The clinical outcome results are summarized in
Similarly, there was a significant increase in GMFM (Rosenbaum, P. L., et al., JAMA 288, 1357-1363 (2002)) scores over the treatment period with a mean improvement of 8.9 (p=0.01). The majority of patients had marked increase in GMFM scores following three months of treatment except patients 2, 7 and 9. Patient 2 had no change in GMFM score following three months of treatment, however, at six months the GMFM score increased from 7.6 to 19.8. Patient 7 had a high level of motor function prior to treatment with a score of 97.5 (maximum possible score 100) that did not change.
On the movement disorder-childhood rating scale (MD-CRS)—a validated measurement of pediatric movement disorders and dystonia—each child had improvement of one class, indicating significant improvement in dystonia and spasticity symptoms (Battini, R., et al., Pediatr Neurol 40, 258-264 (2009); Battini, R., et al., Pediatr Neurol 39, 259-265 (2008)).
Finally, there was a significant improvement (14.6, p=0.03) in quality of life score on the NQL neuromuscular module (Varni, J. W., et al., Ambul Pediatr 3, 329-341 (2003)). This significant improvement is consistent with the improvement observed in section 4 of the NPMDS, which is focused on quality of life.
Biomarker Analysis:
In order to verify the association of Leigh syndrome with decreased levels of reduced glutathione and to validate alpha-tocotrienol quinone's mechanism of action (repletion of reduced glutathione), serial intracellular glutathione levels were obtained on each subject (
Plasma creatine levels were also obtained in all patients. All patients had markedly elevated plasma creatine levels prior to treatment and there was an overall average decrease in creatine of 20.2 points (p=0.15).
Safety and Tolerability:
Treatment with alpha-tocotrienol quinone was not associated with any drug-induced adverse events. There were three serious adverse events due to intercurrent illness during the three month trial (two cases of bronchitis and one case of salmonellosis, all resolved). Other reported adverse conditions included mild sleepiness and hypotonia. There were no abnormalities in clinical chemistry laboratory parameters, including liver and kidney function tests and coagulation parameters. All recorded values were within the normal range for the clinical laboratories used for the analysis.
The initial clinical dose was selected based on laboratory and human patient pharmacokinetic modeling data. Specifically, a three-compartment model for oral administration was employed that included GI absorption delay, as well as one central and one peripheral compartment. A plasma trough drug concentration was targeted 10-20 fold above the ˜10 nM in vitro EC50 value of alpha-tocotrienol quinone that protected Leigh syndrome (SURF1) primary cells against glutathione depletion-mediated apoptotic cell death. Based on serum values of alpha-tocotrienol quinone obtained in two Leigh syndrome subjects after a single 100 mg dose of alpha-tocotrienol quinone was administered, a clinical dose of 100 mg three times daily was calculated and selected as the initial dose. This regimen resulted in an average trough concentration of ˜200 nM at eight hours post dosing. This was consistent with the in vitro target dose and was confirmed in multiple pediatric subjects with inherited mitochondrial disease (Enns, G. M., et al., Mol. Genet. Metab. 105, 91-102 (2012); Blankenberg, F., et al., Ann. Neurol. 70, S148-S148 (2011)). This dose was also safely below the preclinical no observed adverse effect level (NOAEL).
The glutathione cycle was targeted as the translational keystone to link Leigh syndrome pathophysiology and alpha-tocotrienol quinone drug action (Iuso, A., et al., J. Biol. Chem. 281, 10374-10380 (2006)). Specifically, drug action was measured as a function of lymphocyte oxidized and reduced glutathione and total serum thiol content. These blood-based glutathione cycle pharmacodynamic biomarkers were serially measured and correlated with their normalization and, secondarily, with clinical response.
Systemic glutathione status was clinically assessed through serial HPLC measurement of lymphocyte oxidized and reduced glutathione. In addition, total serum thiol content was also determined spectrophotometrically as a measure of total glutathione charge capacity. Each subject was employed as his or her own control. While some inter-patient variability was recorded, all subjects showed a remarkably rapid normalization of glutathione charge, as well as repletion of the total glutathione charge capacity. In several subjects, biochemical response was recorded within two weeks, preceding clinical response. Restoration of glutathione charge was durable in all subjects and continued for all subjects treated >90 days in the extension phase of the protocol. The serum glutathione cycle data reported herein for Leigh syndrome subjects is also consistent with recently reported data on non-invasive measurement of brain glutathione levels using 99-Tc HMPAO SPECT imaging for patients with Leigh syndrome and other mitochondrial diseases treated with alpha-tocotrienol quinone (Enns, G. M., et al., Mol. Genet. Metab. 105, 91-102 (2012); Blankenberg, F., et al., Ann. Neurol. 70, S148-S148 (2011)). Together these data confirm that the instantiated defect in glutathione cycle observed in Leigh syndrome and mitochondrial disease can be detected through blood and/or noninvasive imaging metrics. The data further show that alpha-tocotrienol quinone effects a restoration of glutathione charge and capacity consistent with the pathophysiology of Leigh syndrome and the mechanism of action of alpha-tocotrienol quinone.
To minimize patient-to-patient variability, enrollment was confined to children with the diagnosis of genetically confirmed Leigh syndrome and relatively advanced and progressive stage of disease. Despite these rather narrow selection criteria, there were 10 children in the study who ranged in ages from one to 13. They possessed seven different genetic mutations spanning the respiratory chain and intermediary metabolism. Four outcome measures were used to capture the clinical spectrum of Leigh syndrome and alpha-tocotrienol quinone response to treatment. These included the mitochondrial disease-specific Newcastle Pediatric Mitochondrial Disease score (NPMDS), the more generalized pediatric Gross Motor Function Measure (GMFM), the Movement Disorder-Childhood Rating Scale (MD-CRS) and the Pediatric Quality of Life Inventory (PedsQL). A statistically significant improvement was shown in all four of these outcome measurements. Central nervous system and neuromuscular function improved following treatment with alpha-tocotrienol quinone as assessed by the NPMDS modules 1-3, the GMFM, and the MDCRS. Consistent with improvement in physician-recorded outcome measures, patient families reported improvement in quality of life measures (module 4 of NPMDS and the PedsQL), both of which were statistically significant. Clinical response was not only rapid—within eight weeks—but was universal and durable. Improvement was independent of genotype, age, sex, and disease severity. Additionally, three of the ten subjects have entered the extension phase of the protocol (treatment from 90-180 days). Each of these patients has demonstrated continued improvement in all outcome measurements, suggesting therapeutic durability and indeterminate net clinical benefit.
Given the well-documented natural history of Leigh syndrome (see Table 3,
Example 2 demonstrates that alpha-tocotrienol quinone: i) records a favorable EC50 (rescue) in patient primary cell lines with Leigh syndrome depleted in glutathione; ii) clinically restores the glutathione cycle charge and thiol content; iii) results in neurological and neuromuscular improvement in genetically defined Leigh syndrome subjects independent of genotype; iv) causes disease arrest and reversal, a result which has not previously been recorded in a progressive neurodegenerative disease; and v) can represent a first-in-class drug targeting a first-in-class pathway—the glutathione cycle—for the treatment of Leigh syndrome.
Formulations and AdministrationCompounds and compositions for use in the invention can be prepared as a medicinal preparation or in various other media, such as foods for humans or animals, including medical foods and dietary supplements. A “medical food” is a product that is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements exist (see United States Code, Title 21, Chapter 9, Subchapter V, Part B, Section 360ee). By way of example, but not limitation, medical foods may include vitamin and mineral formulations fed through a feeding tube (referred to as enteral administration). A “dietary supplement” is a product that is intended to supplement the human diet and is typically provided in the form of a pill, capsule, and tablet or like formulation. By way of example, but not limitation, a dietary supplement may include one or more of the following ingredients: vitamins, minerals, herbs, botanicals; amino acids, dietary substances intended to supplement the diet by increasing total dietary intake, and concentrates, metabolites, constituents, extracts or combinations of any of the foregoing. Dietary supplements may also be incorporated into food, including, but not limited to, food bars, beverages, powders, cereals, cooked foods, food additives and candies; or other functional foods designed to promote health, to treat a disease or disorder, to halt the progression of a disease or disorder, or to suppress symptoms of a disease or disorder. If administered as a medicinal preparation, the composition can be administered, either as a prophylaxis or treatment, to a patient in any of a number of methods. The compositions may be administered alone or in combination with other pharmaceutical agents and can be combined with a physiologically acceptable carrier thereof. The effective amount and method of administration of the particular formulation can vary based on the individual subject, the stage of disease or disorder, and other factors evident to one skilled in the art. During the course of the treatment, the concentration of the compounds or compositions may be monitored to insure that the desired level is maintained. The compounds or compositions may be compounded with other physiologically acceptable materials which can be ingested including, but not limited to, foods.
The term “nutraceutical” has been used to refer to any substance that is a food or a part of a food and provides medical or health benefits, including the prevention and treatment of disease. Hence, compositions falling under the label “nutraceutical” may range from isolated nutrients, dietary supplements and specific diets to genetically engineered designer foods, herbal products, and processed foods such as cereals, soups and beverages. In a more technical sense, the term has been used to refer to a product isolated or purified from foods, and generally sold in medicinal forms not usually associated with food and demonstrated to have a physiological benefit or provide protection against chronic disease. Accordingly, the compounds described for use herein can also be administered as nutraceutical or nutritional formulations, with additives such as nutraceutically or nutritionally acceptable excipients, nutraceutically or nutritionally acceptable carriers, and nutraceutically or nutritionally acceptable vehicles. Suitable nutraceutically acceptable excipients may include liquid solutions such as a solution comprising one or more vegetable-derived oils, such as sesame oil, and/or one or more animal-derived oils, and/or one or more fish-derived oils.
Compounds for use in the invention can be formulated as pharmaceutical compositions by formulation with additives such as pharmaceutically acceptable excipients, pharmaceutically acceptable carriers, and pharmaceutically acceptable vehicles. Suitable pharmaceutically acceptable excipients, carriers and vehicles include processing agents and drug delivery modifiers and enhancers, such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and the like, as well as combinations of any two or more thereof. Other suitable pharmaceutically acceptable excipients are described in “Remington's Pharmaceutical Sciences,” Mack Pub. Co., New Jersey (1991), and “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, Philadelphia, 20th edition (2003) and 21st edition (2005), incorporated herein by reference.
A pharmaceutical composition can comprise a unit dose formulation, where the unit dose is a dose sufficient to have a therapeutic or suppressive effect on a disease or disorder or an amount effective to modulate, normalize, or enhance an energy biomarker. The unit dose may be sufficient as a single dose to have a therapeutic or suppressive effect on a disease or disorder, or an amount effective to modulate, normalize, or enhance an energy biomarker. Alternatively, the unit dose may be a dose administered periodically in a course of treatment or suppression of a disease or disorder, or to modulate, normalize, or enhance an energy biomarker.
Pharmaceutical compositions containing the compounds of the invention may be in any form suitable for the intended method of administration, including, for example, a solution, a suspension, or an emulsion. Liquid carriers are typically used in preparing solutions, suspensions, and emulsions. Liquid carriers contemplated for use in the practice of the present invention include, for example, water, saline, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oils or fats, and the like, as well as mixtures of two or more thereof. The liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, stabilizers, and the like. Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols. Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like. For parenteral administration, the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate, and the like. Compositions useful in the present invention may also be in the form of microparticles, microcapsules, liposomal encapsulates, and the like, as well as combinations of any two or more thereof.
Time-release or controlled release delivery systems may be used, such as a diffusion controlled matrix system or an erodible system, as described for example in: Lee, “Diffusion-Controlled Matrix Systems”, pp. 155-198 and Ron and Langer, “Erodible Systems”, pp. 199-224, in “Treatise on Controlled Drug Delivery”, A. Kydonieus Ed., Marcel Dekker, Inc., New York 1992. The matrix may be, for example, a biodegradable material that can degrade spontaneously in situ and in vivo for, example, by hydrolysis or enzymatic cleavage, e.g., by proteases. The delivery system may be, for example, a naturally occurring or synthetic polymer or copolymer, for example in the form of a hydrogel. Exemplary polymers with cleavable linkages include polyesters, polyorthoesters, polyanhydrides, polysaccharides, poly(phosphoesters), polyamides, polyurethanes, poly(imidocarbonates) and poly(phosphazenes).
The compounds used in the methods of the invention may be administered to the individual in any suitable form that will provide a sufficient concentration of the compounds in the cell(s), tissue(s) or body compartment(s) of interest, such as in the plasma and/or central nervous system levels of the individual. Compounds for use in the invention may be administered enterally, orally, parenterally, sublingually, by inhalation (e.g. as mists or sprays), rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. For example, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoretic, intravenous, intraarterial, intramuscular, intraperitoneal, intranasal (e.g. via nasal mucosa), subdural, rectal, vaginal, gastrointestinal, and the like, and directly to a specific or affected organ or tissue. For delivery to the central nervous system, spinal and epidural administration, or administration to cerebral ventricles, can be used. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous injection, intraarterial injection, intramuscular injection, intrasternal injection, or infusion techniques. The compounds are mixed with pharmaceutically acceptable carriers, adjuvants, and vehicles appropriate for the desired route of administration. Oral administration is a preferred route of administration, and formulations suitable for oral administration are preferred formulations. Oral administration is advantageous due to its ease of implementation and patient (or caretaker) compliance. For patients with difficulty in swallowing, introduction of medicine via feeding tube, feeding syringe or gastrostomy can be employed in order to accomplish enteric administration. The active compound (and, if present, other co-administered agents) can be enterally administered in sesame oil, or any other pharmaceutically acceptable carrier suitable for formulation for administration via feeding tube, feeding syringe, or gastrostomy. Compounds for use in the invention can be administered in solid form, in liquid form, in aerosol form, or in the form of tablets, pills, powder mixtures, capsules, granules, injectables, creams, solutions, suppositories, enemas, colonic irrigations, emulsions, dispersions, food premixes, and in other suitable forms. The compounds can also be administered in liposome formulations. The compounds can also be administered as prodrugs, where the prodrug undergoes transformation in the treated subject to a form which is therapeutically effective. Additional methods of administration are known in the art.
If administration to the central nervous system is desired, it is preferable to administer compounds that cross the blood-brain barrier. However, compounds that do not cross the blood-brain barrier can be delivered to the central nervous system by spinal and epidural administration, or administration to cerebral ventricles.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in propylene glycol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
In certain embodiments of the invention, especially those embodiments where a formulation is used for injection or other parenteral administration including the routes listed herein, but also including embodiments used for oral, gastric, gastrointestinal, enteric, or other routes of administration, the formulations and preparations used in the methods of the invention are sterile. Sterile pharmaceutical formulations are compounded or manufactured according to pharmaceutical-grade sterilization standards (United States Pharmacopeia Chapters 797, 1072, and 1211; California Business & Professions Code 4127.7; 16 California Code of Regulations 1751, 21 Code of Federal Regulations 211) known to those of skill in the art.
Suppositories for rectal administration of a compound can be prepared by mixing the compound with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols that are solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum and release the compound.
Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents. Alternatively, the compound may also be administered in neat form if suitable.
Compounds useful in the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y., p. 33 et seq (1976).
The invention also provides articles of manufacture and kits containing materials useful for treating, preventing or suppressing symptoms associated with a disease or disorder, or modulating, normalizing, or enhancing an energy biomarker. The article of manufacture comprises a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition having an active agent which is effective for treating, preventing or suppressing symptoms associated with a disease or disorder, or modulating, normalizing, or enhancing an energy biomarker. The active agent in the composition is one or more of the compounds of the invention. The label on the container indicates that the composition is used for treating, preventing or suppressing symptoms associated with a disease or disorder, or modulating, normalizing, or enhancing an energy biomarker, and may also indicate directions for either in vivo or in vitro use, such as those described above.
The invention also provides kits comprising any one or more of the compounds of the invention. In some embodiments, the kit of the invention comprises the container described above. In other embodiments, the kit of the invention comprises the container described above and a second container comprising a buffer. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein.
In other aspects, the kits may be used for any of the methods described herein, including, for example, to treat an individual with symptoms associated with a disease or disorder, to prevent symptoms associated with a disease or disorder, or to suppress symptoms associated with a disease or disorder in an individual, or for modulating, normalizing, or enhancing an energy biomarker.
The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host to which the active ingredient is administered and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, body area, body mass index (BMI), general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the type, progression, and severity of the particular disease or disorder undergoing therapy. The unit dosage chosen is usually fabricated and administered to provide a defined final concentration of drug in the blood, cerebrospinal fluid, brain tissues, spinal cord tissues, other tissues, other organs, or other targeted region of the body. The effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.
Examples of dosages which can be used are an effective amount of compounds within the dosage range of about 0.1 μg/kg to about 300 mg/kg body weight, or within about 0.1 mg/kg to about 300 mg/kg body weight, or within about 1.0 μg/kg to about 40 mg/kg body weight, or within about 1.0 μg/kg to about 20 mg/kg body weight, or within about 1.0 μg/kg to about 10 mg/kg body weight, or within about 10.0 μg/kg to about 10 mg/kg body weight, or within about 100 μg/kg to about 10 mg/kg body weight, or within about 0.1 mg/kg to about 100 mg/kg body weight, or within about 0.1 mg/kg to about 80 mg/kg body weight, or within about 0.1 mg/kg to about 50 mg/kg body weight, or within about 0.1 mg/kg to about 30 mg/kg body weight, or within about 1.0 mg/kg to about 80 mg/kg body weight, or within about 1.0 mg/kg to about 50 mg/kg body weight, or within about 1.0 mg/kg to about 30 mg/kg body weight, or within about 1.0 mg/kg to about 10 mg/kg body weight, or within about 10 mg/kg to about 100 mg/kg body weight, or within about 10 mg/kg to about 80 mg/kg body weight, or within about 50 mg/kg to about 150 mg/kg body weight, or within about 100 mg/kg to about 200 mg/kg body weight, or within about 150 mg/kg to about 250 mg/kg body weight, or within about 200 mg/kg to about 300 mg/kg body weight, or within about 250 mg/kg to about 300 mg/kg body weight. Other dosages which can be used are about 0.01 mg/kg body weight, about 0.1 mg/kg body weight, about 1 mg/kg body weight, about 10 mg/kg body weight, about 20 mg/kg body weight, about 30 mg/kg body weight, about 40 mg/kg body weight, about 50 mg/kg body weight, about 75 mg/kg body weight, about 100 mg/kg body weight, about 125 mg/kg body weight, about 150 mg/kg body weight, about 175 mg/kg body weight, about 200 mg/kg body weight, about 225 mg/kg body weight, about 250 mg/kg body weight, about 275 mg/kg body weight, or about 300 mg/kg body weight, or about 0.1, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 mg total. Compounds useful in the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided dosage of two, three or four times daily. These dosages can be administered for a short term, such as days or weeks, or for a long term, for example, over months, years, or even over the entire lifetime of the patient.
While the compounds useful in the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other agents used in the treatment or suppression of diseases or disorders.
When additional active agents are used in combination with compounds useful in the present invention, the additional active agents may generally be employed in therapeutic amounts as indicated in the Physicians' Desk Reference (PDR) 53rd Edition (1999), which is incorporated herein by reference, or such therapeutically useful amounts as would be known to one of ordinary skill in the art.
Compounds useful in the invention and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compounds in the compositions of the invention may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. When administered in combination with other therapeutic agents, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
The particular dosage appropriate for a specific patient is determined by dose titration. The starting dose can be estimated based on the United States Food and Drug Administration guidelines titled “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” (July 2005) as well as the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines titled “Guidance on Non-clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals” (July 2008). Per ICH guidelines, predicted exposures from the starting dose should not exceed 1/50th the NOAEL (No-Adverse-Observed-Effect-Level) in the more sensitive species on a mg/m2 basis.
Additional Agents for Co-Administration with Compounds Useful in the Invention
Representative additional agents useful in combination with the compounds of the invention for the treatment, prevention or suppression of mitochondrial diseases include, but are not limited to, Coenzyme Q, vitamin E, idebenone, MitoQ, vitamins, and antioxidant compounds.
One particularly advantageous additional agent for co-administration is N-acetyl cysteine (NAC), which is a precursor to glutathione. About 50 mg to about 1200 mg of NAC, about 50 mg to about 1000 mg of NAC, about 300 mg to about 1200 mg of NAC, or about 300 mg to about 1000 mg of NAC can be co-administered with compounds useful in the invention on a daily basis. This is particularly useful for individuals with low total glutathione levels, such as individuals with a total serum glutathione level below about 30 nmol/mg cell protein, or individuals with a total serum glutathione level below about 1 micromolar.
The co-administered agents can be administered simultaneously with, prior to, or after, administration of the primary compound.
KitsThe invention also provides articles of manufacture and kits containing materials. The article of manufacture comprises a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The kit may also contain directions for use in treatment.
The invention also provides kits comprising any one or more of a compound selected from alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocotrienol hydroquinone, beta-tocotrienol hydroquinone, gamma-tocotrienol hydroquinone, and delta-tocotrienol hydroquinone, or a composition comprising an active agent selected from alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocotrienol hydroquinone, beta-tocotrienol hydroquinone, gamma-tocotrienol hydroquinone, and delta-tocotrienol hydroquinone. In some embodiments, the kit of the invention comprises the container described above, which holds a compound selected from alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocotrienol hydroquinone, beta-tocotrienol hydroquinone, gamma-tocotrienol hydroquinone, and delta-tocotrienol hydroquinone, or a composition comprising an active agent selected from alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocotrienol hydroquinone, beta-tocotrienol hydroquinone, gamma-tocotrienol hydroquinone, and delta-tocotrienol hydroquinone. In other embodiments, the kit of the invention comprises the container described above, which holds a compound selected from alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocotrienol hydroquinone, beta-tocotrienol hydroquinone, gamma-tocotrienol hydroquinone, and delta-tocotrienol hydroquinone, or a composition comprising an active agent selected from alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocotrienol hydroquinone, beta-tocotrienol hydroquinone, gamma-tocotrienol hydroquinone, and delta-tocotrienol hydroquinone, and a second container comprising a vehicle for the compound or composition, such as one or more vegetable-derived oils, such as sesame oil, and/or one or more animal-derived oils, and/or one or more fish-derived oils. In other embodiments, the kit of the invention comprises the container described above, which holds a compound selected from alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocotrienol hydroquinone, beta-tocotrienol hydroquinone, gamma-tocotrienol hydroquinone, and delta-tocotrienol hydroquinone, or a composition comprising an active agent selected from alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocotrienol hydroquinone, beta-tocotrienol hydroquinone, gamma-tocotrienol hydroquinone, and delta-tocotrienol hydroquinone, where the compound or composition has been pre-mixed with a vehicle for the compound or composition, such as one or more vegetable-derived oils, such as sesame oil, and/or one or more animal-derived oils, and/or one or more fish-derived oils. The kits may further include other materials desirable from a commercial and user standpoint, including other vehicles, buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any of the methods described herein.
In other aspects, the kits may be used for any of the methods described herein.
The invention will be further understood by the following nonlimiting examples.
EXAMPLES Example 1 HPLC Determination of Free GSH, Protein Bound GSH (GS-Pro) and GSSG in LymphocytesThe determination of the different forms of GSH obtained from lymphocytes can be performed in a manner similar to the method previously reported by Pastore et al. (Clin. Chem. (Washington, D.C.) 44, 825-832 (1998)). Briefly, 30 μl of 4 M NaBH4, 20 μl of 2 mM EDTA/DTT, 10 μl of 1-octanol and 20 μl of 1.8 M HCl are placed in the derivatization vial containing 30 μl of sample. After the mixture is incubated for three min, 100 μl of 1.5 M N-ethylmorpholine buffer, pH 8.0, 400 μl of distilled water, and 20 μl of 25 mM bromobimane are added. After an additional three-min incubation, 40 μl of acetic acid are added and 20 μl (for free GSH) or 80 μl (for GSSG and GS-Pro) of this mixture are injected into the column. The thiol derivatives are quantified by HPLC (Agilent Technologies 1100 HPLC with a fluorescence detector-excitation at 390 nm and an emission at 478 nm). The analytes are separated for detection using a C18 column (Hypersil ODS; 150×4.6 mm, 3 micron) at 1.5 ml/min using an aqueous phase of aqueous 30 mM ammonium nitrate and 40 mM ammonium formate at pH 3.6 with an increasing acetonitrile gradient. HPLC determinations of the various forms of GSH are carried out on lymphocytes sonicated three times for two sec in 0.1 ml of 0.1 M potassium-phosphate buffer, pH 7.2 using a Model VC 130 Vibra Cell™ ultrasonic processor. For free GSH determinations, 100 μl of 12% sulfosalicylic acid are added to 50 μl of cells lysate, and analytes content on the acid-soluble fraction are determined. The protein pellet is dissolved in 150 μl of 0.1 N NaOH, and protein bound glutathione (GS-Pro) determined. For GSSG determination, cells are sonicated in the presence of 5 mM N-ethylmaleimide (NEM); 100 μl of 12% sulfosalicylic acid are added to 50 μl homogenates, and analytes content on the acid-soluble fraction are determined. Protein concentrations are quantified by BCA-protein assay.
Example 2 Modulation of Glutathione Levels in Treatment of Leigh DiseaseStudy Overview:
A prospective single arm subject-controlled trial of alpha-tocotrienol quinone was performed in children with genetically-confirmed Leigh syndrome (Table 1,
Subjects:
Subjects were children with a genetically confirmed diagnosis of Leigh syndrome who had a Newcastle Pediatric Mitochondrial Disease Scale (NPMDS) on sections 1-3 greater than 15—signifying at least moderately severe disease progression. All participants were required to have MRI confirmation of necrotizing encephalopathy. In addition, all children were required to discontinue the use of CoQ10 and any other antioxidant supplements for the duration of the trial. Informed consent was obtained from the parents of each child.
Intervention:
All subjects received 100 mg of alpha-tocotrienol quinone three times daily orally or via gastrostomy tube. Total treatment length was three months, with an extension phase for children who completed the initial treatment period. All adverse and serious adverse events were tracked in the trial database. In addition, all held doses due to intolerance of alpha-tocotrienol quinone or any other reason were also recorded.
Endpoints—Clinical Endpoints:
The primary endpoints of this study were the Newcastle Pediatric Mitochondrial Disease Scale (NPMDS), the Gross Motor Function Measure (GMFM-66 item) and the PedsQL Neuromuscular Module (PedsQL). The NPDMS is a scale developed for and validated in children with inherited mitochondrial diseases to assess disease severity and progression. Sections 1-3 of the NPDMS assess organ specific function and section 4 is a quality of life assessment. In this study, serial NPMDS measurements were utilized to assess alpha-tocotrienol quinone effect on disease progression. The GMFM is an observational tool used to assess gross motor function over time in children with neuromuscular disorders. The GMFM has been validated as an outcome measurement instrument in the intervention trials of children with neuromuscular disorders. The PedsQL is a validated measurement of pediatric quality of life and the neuromuscular module utilized in this study is specific to children with neuromuscular disorders. In addition, clinical response was measured using the Movement-Disorder-Childhood Rating Scale (MD-CRS), a validated instrument for assessing dystonia and spasticity in children. Results are shown in
Biomarker Endpoints:
Intracellular reduced glutathione (GSH) serves as the cell's principal endogenous antioxidant. Leigh syndrome and other mitochondrial diseases are associated with decreased GSH levels as the high level of oxidative stress and free oxygen radicals deplete cellular GSH reserves. In order to verify alpha-tocotrienol quinone mechanism of action and to correlate this action with clinical benefit, intracellular (lymphocyte) glutathione levels were measured in all patients. The ratio of recued-to-oxidized glutathione and reduced glutathione to total glutathione levels were calculated to assess the effect of alpha-tocotrienol quinone on repleting reduced intracellular glutathione. Results are shown in
Analytical Methods:
For lymphocytes and erythrocyte isolation from whole blood, venous blood samples were layered on the top of a discontinuous gradient consisting of 3 ml of Hystopaque®-1119 and 3 mL of Hystopaque®-1077. Gradients were centrifuged at 750 g for 40 min at 4° C. The lymphocyte layer was transferred to a 15 ml centrifuge tube, diluted to a volume of 10 ml with 0.9% NaCl and centrifuged at 750 g for 40 min. The supernatant was removed and, if necessary, the lysis of erythrocytes was performed by adding 2 ml H2O. After 90 s, 2 ml of 1.8% NaCl solution was added and cells washed four times with 0.9% NaCl. The pellet of lymphocytes was centrifuged at 750 g for 10 min and stored at −80° C. until HPLC analysis.
HPLC Determination of Free GSH, Protein Bound GSH and GSSG in Lymphocytes:
The determination of the different forms of GSH obtained from lymphocytes was performed, with little modification to a method previously reported (Pastore, A., et al. Clin. Chem. (Washington, D.C.) 44, 825-832 (1998)). Briefly, 30 μl of 4 M NaBH4, 20 μl of 2 mM EDTA/DTT, 10 μl of 1-octanol and 20 μl of 1.8 M HCl were placed in the derivatization vial containing 30 μl of sample. After the mixture was incubated for three min, 100 μl of 1.5 M N-ethylmorpholine buffer, pH 8.0, 400 μl of distilled water, and 20 μl of 25 mM bromobimane were added. After an additional three-min incubation, 40 μl of acetic acid was added and 20 μl (for free GSH) or 80 μl (for GSSG and GS-Pro) of this mixture was injected into the column. The thiol derivatives were quantified by HPLC (Agilent Technologies 1100 HPLC with a fluorescence detector-excitation at 390 nm and an emission at 478 nm). The analytes were separated for detection using a C18 column (Hypersil ODS; 150×4.6 mm, 3 micron) at 1.5 ml/min using an aqueous phase of aqueous 30 mM ammonium nitrate and 40 mM ammonium formate at pH 3.6 with an increasing acetonitrile gradient. HPLC determinations of the various forms of GSH were carried out on lymphocytes sonicated three times for two sec in 0.1 ml of 0.1 M potassium-phosphate buffer, pH 7.2 using a Model VC 130 Vibra Cell™ ultrasonic processor. For free GSH determinations, 100 μl of 12% sulfosalicylic acid were added to 50 μl of cells lysate, and analytes content on the acid-soluble fraction was determined. The protein pellet was dissolved in 150 μl of 0.1 N NaOH, and protein bound glutathione (GS-Pro) determined. For GSSG determination, cells were sonicated in the presence of 5 mM N-ethylmaleimide (NEM); 100 μl of 12% sulfosalicylic acid were added to 50 μl homogenates, and analytes content on the acid-soluble fraction was determined. Protein concentrations were quantified by BCA-protein assay.
Total Free Thiols in Plasma:
Total thiols were determined in plasma following the method reported by M.-L. Hu (Methods Enzymol. 233, 380-385 (1994)). 50 μl plasma was mixed with 1 ml of the Tris-EDTA buffer and the absorbance at 412 nm was measured. Then, 20 μl of 10 mM DTNB were added. After 15 min incubation at room temperature, the absorption at 412 nm (ε=13,600 cm−1M−1) was measured together with a DTNB blank.
Statistical Analyses:
Descriptive statistics were performed on the entire cohort of children enrolled in this study. Changes in outcome measurement were calculated for each subject as a proportion of their baseline level. Overall mean changes from baseline were analyzed using a Wilcoxon signed rank test. Statistical significance defined as p≦0.05. For patients who entered into the extension phase of the study, data are reported but not included in the calculation of mean change from baseline.
Results of the study are shown in Table 2, demonstrating that treatment with EPI-743 resulted in a significant enhancement of levels of reduced glutathione and a reduction in oxidized glutathione.
The invention is described further by the following embodiments.
Embodiment 1A method of treating a subject having a glutathione redox potential disorder, comprising the steps of: a) altering the redox potential of glutathione in the subject by administering a compound to the subject at an initial dosage level that alters the concentration of reduced glutathione in the subject, the concentration of oxidized glutathione in the subject, or both the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject; b) subsequent to administering the compound to the subject, measuring the concentration of reduced glutathione and oxidized glutathione in the subject; c) calculating the redox potential of glutathione in the subject; d) adjusting the dosage of the compound administered to the subject; and e) repeating steps b), c), and d) until a ratio of oxidized glutathione concentration to reduced glutathione concentration between about 0.01 and about 0.5 is attained.
Embodiment 2A method of treating a subject having a glutathione redox potential disorder, comprising the steps of: a) altering the redox potential of glutathione in the subject by administering a compound to the subject at an initial dosage level that alters the concentration of reduced glutathione in the subject, the concentration of oxidized glutathione in the subject, or both the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject; b) subsequent to administering the compound to the subject, measuring the concentration of reduced glutathione and oxidized glutathione in the subject; c) calculating the redox potential of glutathione in the subject; d) adjusting the dosage of the compound administered to the subject; and e) repeating steps b), c), and d) until the subject has a redox potential of at least about an absolute value of 10 mV more negative than the redox potential of the subject prior to treatment.
Embodiment 3The method of Embodiment 1, wherein the subject having a glutathione redox potential disorder has a ratio of oxidized glutathione concentration to reduced glutathione concentration of about 2 or greater prior to administering the compound.
Embodiment 4The method of any of Embodiments 1-3, wherein the step of measuring the concentration of reduced glutathione and the concentration of oxidized glutathione comprises an HPLC measurement.
Embodiment 5The method of any of Embodiments 1-4, wherein the step of measuring the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject comprises measuring the concentration of reduced glutathione and the concentration of oxidized glutathione in the blood plasma, blood serum, whole blood, cerebrospinal fluid, semen, breast milk, umbilical cord blood, umbilical cord tissue, or skin biopsy of the subject.
Embodiment 6The method of any of Embodiments 1-4, wherein the step of measuring the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject comprises measuring the concentration of reduced glutathione and the concentration of oxidized glutathione in the lymphocytes of the subject.
Embodiment 7The method of any of Embodiments 1-7, wherein step a) and step d) additionally comprise measuring the concentration of total glutathione in the subject, or calculating the concentration of total glutathione in the subject from the measured concentration of reduced glutathione and concentration of oxidized glutathione in the subject, and step a) additionally comprises administering a therapeutically effective amount of N-acetyl cysteine to the subject if the concentration of total glutathione in the subject is below about 30 nmol/mg cell protein, or if the concentration of total glutathione in the subject is below about 1 micromolar.
Embodiment 8The method of Embodiment 7, wherein sufficient N-acetyl cysteine is administered to the subject to adjust the concentration of total glutathione in the subject above about 30 nmol/mg cell protein, or to adjust the concentration of total glutathione in the subject above about 1 micromolar.
Embodiment 9The method of Embodiment 7, wherein a dosage of about 50 mg to about 1000 mg N-acetyl cysteine is administered to the subject.
Embodiment 10The method of any of Embodiments 1-9, wherein the compound administered to the subject that alters the concentration of reduced glutathione in the subject, the concentration of oxidized glutathione in the subject, or both the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject is selected from the group consisting of pharmaceutically acceptable two-electron redox-active molecules having a reduction potential between about −350 millivolts and about +150 millivolts versus the standard hydrogen electrode.
Embodiment 11The method of any of Embodiments 1-9, wherein the compound administered to the subject that alters the concentration of reduced glutathione in the subject, the concentration of oxidized glutathione in the subject, or both the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject is selected from the group consisting of pharmaceutically acceptable two-electron redox-active molecules having a reduction potential between about 20 millivolts more reductive than Coenzyme Q and about 250 millivolts more reductive than Coenzyme Q.
Embodiment 12The method of any of Embodiments 1-9, wherein the compound administered to the subject that alters the concentration of reduced glutathione in the subject, the concentration of oxidized glutathione in the subject, or both the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject is selected from the group consisting of compounds of Formula I:
where R is selected from:
where the asterisk * indicates the attachment of R in Formula I; where R1, R2, and R3 are independently H or C1-C6 alkyl, m is an integer from 0 to 9 inclusive, and the bonds indicated by a dashed line can be either double or single bonds.
Embodiment 13The method of any of Embodiments 1-9, wherein the compound administered to the subject is selected from the group consisting of compounds of Formula I-ox or Formula I-red:
where R1, R2, and R3 are independently H or C1-C6 alkyl; m is an integer from 0 to 9 inclusive; and the bonds indicated by a dashed line can be either double or single bonds.
Embodiment 14The method of Embodiment 13, wherein the compound administered to the subject that alters the concentration of reduced glutathione in the subject, the concentration of oxidized glutathione in the subject, or both the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject is selected from the group consisting of: alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocopherol quinone, beta-tocopherol quinone, gamma-tocopherol quinone, and delta-tocopherol quinone, or any combination thereof.
The disclosures of all publications, patents, patent applications and published patent applications referred to herein by an identifying citation are hereby incorporated herein by reference in their entirety.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is apparent to those skilled in the art that certain minor changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention.
Claims
1. A method of treating a subject having a glutathione redox potential disorder, comprising the steps of:
- a) altering the redox potential of glutathione in the subject by administering a compound to the subject at an initial dosage level that alters the concentration of reduced glutathione in the subject, the concentration of oxidized glutathione in the subject, or both the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject;
- b) subsequent to administering the compound to the subject, measuring the concentration of reduced glutathione and oxidized glutathione in the subject;
- c) calculating the redox potential of glutathione in the subject;
- d) adjusting the dosage of the compound administered to the subject; and
- e) repeating steps b), c), and d) until a ratio of oxidized glutathione concentration to reduced glutathione concentration between about 0.01 and about 0.5 is attained.
2. A method of treating a subject having a glutathione redox potential disorder, comprising the steps of:
- a) altering the redox potential of glutathione in the subject by administering a compound to the subject at an initial dosage level that alters the concentration of reduced glutathione in the subject, the concentration of oxidized glutathione in the subject, or both the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject;
- b) subsequent to administering the compound to the subject, measuring the concentration of reduced glutathione and oxidized glutathione in the subject;
- c) calculating the redox potential of glutathione in the subject;
- d) adjusting the dosage of the compound administered to the subject; and
- e) repeating steps b), c), and d) until the redox potential of glutathione in the subject is at least an absolute value of about 10 mV more negative than the redox potential of glutathione in the subject prior to treatment.
3. The method of claim 1, wherein the subject having a glutathione redox potential disorder has a ratio of oxidized glutathione concentration to reduced glutathione concentration of about 2 or greater prior to administering the compound.
4. The method of claim 1, wherein the step of measuring the concentration of reduced glutathione and the concentration of oxidized glutathione comprises an HPLC measurement.
5. The method of claim 1, wherein the step of measuring the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject comprises measuring the concentration of reduced glutathione and the concentration of oxidized glutathione in the blood plasma, blood serum, or cerebrospinal fluid of the subject.
6. The method of claim 1, wherein the step of measuring the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject comprises measuring the concentration of reduced glutathione and the concentration of oxidized glutathione in the lymphocytes of the subject.
7. The method of claim 1, wherein step a) and step d) additionally comprise measuring the concentration of total glutathione in the subject, or calculating the concentration of total glutathione in the subject from the measured concentration of reduced glutathione and concentration of oxidized glutathione in the subject, and step a) additionally comprises administering a therapeutically effective amount of N-acetyl cysteine to the subject if the concentration of total glutathione in the subject is below about 30 nmol/mg cell protein.
8. The method of claim 7, wherein sufficient N-acetyl cysteine is administered to the subject to adjust the concentration of total glutathione in the subject above about 30 nmol/mg cell protein.
9. The method of claim 2, wherein step a) and step d) additionally comprise measuring the concentration of total glutathione in the subject, or calculating the concentration of total glutathione in the subject from the measured concentration of reduced glutathione and concentration of oxidized glutathione in the subject, and step a) additionally comprises administering a therapeutically effective amount of N-acetyl cysteine to the subject if the concentration of total glutathione in the subject is below about 1 micromolar.
10. The method of claim 9, wherein the amount of N-acetyl cysteine administered to the subject is about 50 mg to about 1000 mg.
11. The method of claim 1, wherein the compound administered to the subject that alters the concentration of reduced glutathione in the subject, the concentration of oxidized glutathione in the subject, or both the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject is selected from the group consisting of pharmaceutically acceptable two-electron redox-active molecules having a reduction potential between about −350 millivolts and about +150 millivolts versus the standard hydrogen electrode.
12. The method of claim 1, wherein the compound administered to the subject that alters the concentration of reduced glutathione in the subject, the concentration of oxidized glutathione in the subject, or both the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject is selected from the group consisting of pharmaceutically acceptable two-electron redox-active molecules having a reduction potential between about 20 millivolts more reductive than Coenzyme Q and about 250 millivolts more reductive than Coenzyme Q.
13. The method of claim 1, wherein the compound administered to the subject that alters the concentration of reduced glutathione in the subject, the concentration of oxidized glutathione in the subject, or both the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject is selected from the group consisting of compounds of Formula I:
- where R is selected from:
- where the asterisk * indicates the attachment of R in Formula I;
- where R1, R2, and R3 are independently H or C1-C6 alkyl,
- m is an integer from 0 to 9 inclusive,
- and the bonds indicated by a dashed line can be either double or single bonds.
14. The method of claim 13, wherein the compound administered to the subject is selected from the group consisting of compounds of Formula I-ox or Formula I-red:
- where R1, R2, and R3 are independently H or C1-C6 alkyl;
- m is an integer from 0 to 9 inclusive;
- and the bonds indicated by a dashed line can be either double or single bonds.
15. The method of claim 14, wherein the compound administered to the subject that alters the concentration of reduced glutathione in the subject, the concentration of oxidized glutathione in the subject, or both the concentration of reduced glutathione and the concentration of oxidized glutathione in the subject is selected from the group consisting of: alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocopherol quinone, beta-tocopherol quinone, gamma-tocopherol quinone, and delta-tocopherol quinone, or any combination thereof.
Type: Application
Filed: Sep 6, 2013
Publication Date: Aug 6, 2015
Inventors: Guy M. Miller (Monte Sereno, CA), William D. Shrader (Belmont, CA), Viktoria Kheifets (Mountain View, CA)
Application Number: 14/426,130
